Extracellular pH has been shown to affect cell physiology, though its effect is not as widely studied as other physical parameters like temperature. Unless controlled, pH of a cell culture process can vary during the course of culture, and usually decreases due to formation of waste metabolites like organic acids.
Several references point out that commercial cell culture processes such as for recombinant protein production are affected by pH. pH is typically controlled in bioreactors cultivating animal cell cultures between 6.8 to 7.15 via measurement and subsequent base/CO2 addition, and has been automated in commercially available scale down micro bioreactors (e.g Simcell, Seahorse Bioscience, MA, USA). However, pH is not controlled in widely used small scale culture platforms like shake flasks for suspension cultures due to lack of simple methods, though robotic instrumentation is available commercially for the same (e.g. DASGIP (Germany) and TAP (UK)).
Lack of pH control can result in sub optimal screening due to differences in performance under controlled and uncontrolled pH conditions. Bareither and Pollard (Biotechnol. Prog. 2011; 27:2-14) have recently reviewed small-scale parallel culture formats for accelerated process development and noted that: “The simplified systems of shake flasks and microtiter plates provide significant high throughput capability. Yet they carry less instrumentation which limits the opportunity for data quality and quantity. For this reason they remain as screening tools without implementation of robust pH and DO (dissolved oxygen) control”. To circumvent this, scale down microbioreactors have been recently commercially introduced which are able to control pH of mammalian cell cultures via measurement and subsequent base/CO2 addition (e.g. Ambr™, TAP Biosystems, UK which was demonstrated to maintain pH within ±0.2 units from the set point 7). However, the said pH control involves investment in additional infrastructure.
Zhou et al (Biotechnol. Prog. 2010; 26: 872-880) have reported using SNARF-4F 5-(-and 6)-carboxylic acid pH monitoring for multiwell plates and shake flasks, followed by calculations of required base addition based on a mathematical model, which also needs instrumentation for continuous control.
Further pH is an important parameter that can affect microbial cell growth. pH can decrease significantly during culture in the presence of carbon sources like glucose due to overflow metabolism and a change in pH beyond the permissive range of the organism affects cell growth. It is estimated that more than 90% of all cultures in biotechnology are carried out in shake flasks which traditionally have no control over pH. Decrease in pH of the culture can thus contribute to limited cell densities achieved in shake flasks. To prevent excessive pH drifts, culture medium can be supplemented with high buffer concentrations. However, high buffer concentrations result in increased osmolarity of the culture medium and may be inhibitory to the growth of the micro-organism. Moreover when the amount of protons produced exceeds the capacity of the buffer, the buffer will no longer be able to prevent changes in pH.
Scheidle et al. (BMC Biotechnol. 2011; 11: 25) have recently demonstrated a system releasing sodium carbonate for pH control in a microbial culture for 14 hours, wherein polymer-based controlled-release discs embedding sodium carbonate crystals are used for controlling the pH in shake flasks enabled the successful cultivation of E. coli K12 and E. coli BL21 pRSET eYFP-IL6 in mineral media with glycerol and glucose as carbon sources, respectively.
US2009190135 (Clarizia Lisa et al.) discloses method for maintaining an optimal cell culture pH, comprising providing a pH-sensitive hydrogel comprising a pH-regulating agent or buffer to the cell culture that comprises mouse embryonic stein cells under conditions, such that the regulating agent or buffer is released into the cell culture in response to a change in the pH of the cell culture, such that the optimal cell culture pH is maintained. Also it discloses the autoregulatory nature of the hydrogels' activity which leads to the periodic release of base, maintaining the pH within or very close to the optimal pH range for the cell culture for up to 4 days, refer FIG. 9. However there is no teaching about microbial pH control.
Chen A, Biotechnol. Bioeng. 2009; 102: 148-160, reported Twenty-four well plate miniature bioreactor system as a scale-down model for cell culture process development but suffer from economic disadvantages.
In view of the prior art pH control via measurement and base addition is not easily possible in small-scale culture formats like tissue-culture flasks and shake flasks.
Hydrogels are hydrophilic polymers that absorb water- and are insoluble in water under physiologic conditions due to the existence of a three-dimensional network. Also the application of hydrogels has been widely reported for controlled drug release and tissue engineering.
Therefore the inventors have developed a hydrogel based system loaded with a base for in situ pH maintenance.
The non-maintenance of pH in the desired narrow range also prevents shake flasks to precisely and accurately mimic bioreactor based operation for initial screening during cell line and process development for recombinant protein production in mammalian cells and other such similar operations.
These hydrogels can also be suitably modified with reduced cross-linking and increased surface area for application to faster growing microbial cultures.